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Abstract:

The methods provided herein are based on the discovery, as described in
more detail herein, that aspartyl aminopeptidase (DAP) is expressed in
mammalian pancreatic islet cells and that increased DAP expression
results in decreased angiotensin II (Ang II) levels in the islet cells.
Methods are provided for identifying compounds effective to increase the
expression and/or activity of DAP in mammalian pancreatic islet cells and
thereby are effective to decrease Ang II levels in the islet cells.
Methods are also provided for decreasing Ang II levels in a subject
having Ang II induced pancreas islet cell dysfunction and/or suffering or
susceptible to type 1 diabetes, as well as for the treatment or
prevention of type 1 diabetes comprising administering to the subject a
therapeutically effective amount of the compound identified according to
the methods herein.

Claims:

1. A method for identifying a compound effective to increase aspartyl
aminopeptidase (DAP) activity in mammalian pancreatic islet cells, the
method comprising: contacting DAP and angiotensin II with a test
compound; measuring the level of angiotensin II; and determining if the
level of angiotensin II is lower when treated with the test compound than
an otherwise identical control method containing DAP and angiotensin II
that has not been contacted with the test compound.

2. The method according to claim 1, wherein the contacting step is
carried out at a temperature of about 35.degree. to about 39.degree. C.
in an aqueous solution.

3. The method according to claim 2, wherein the aqueous solution has a pH
of about 7.2 to about 7.6.

4. The method according to claim 1, wherein the method further comprises
measuring activity of DAP and determining if the activity of DAP is
higher when treated with the test compound than an otherwise identical
control containing DAP and angiotensin II that has not been contacted
with the test compound.

5. The method according to claim 1, wherein the DAP and angiotensin II
are contacted with the test compound for an amount of time effective for
the DAP to degrade at least about 10 percent of the angiotensin II.

6. The method according to claim 1, wherein the DAP and angiotensin II
are each in substantially purified form.

7. A method for identifying a compound effective to increase DAP
expression in mammalian pancreatic islet cells, the method comprising:
culturing mammalian islet cells capable of producing DAP in the presence
of a test compound; measuring the level of expression from the gene
encoding DAP; and determining if the level of expression is higher when
treated with the test compound than an otherwise identical control method
with cells not cultured with the test compound.

8. The method according to claim 7, wherein the method further comprises
measuring an amount of angiotensin II and determining if the level of
angiotensin II is lower when treated with the test compound than an
otherwise identical control not cultured with the test compound.

9. The method according to claim 7, wherein measuring the level of
expression comprises measuring the amount of DAP in the cultured cells.

10. The method according to claim 7, wherein measuring the level of
expression comprises measuring the amount of mRNA encoding DAP in the
cultured cells.

11. A method for increasing DAP expression and decreasing Ang II levels
in a subject, the method comprising delivering one or more copies of a
polynucleotide encoding DAP to the subject's pancreatic islet cells.

12. The method according to claim 11, wherein the one or more copies of a
polynucleotide encoding DAP are delivered to the subject's pancreatic
islet cells via a viral vector.

13. The method according to claim 12, wherein the viral vector includes a
promoter activated in pancreatic alpha cells.

14. The method according to claim 12, wherein the promoter is a
glucagon-activated promoter.

15. The method according to claim 11, wherein the polynucleotide encoding
DAP comprises a sequence encoding a protein a having at least about 80
percent homology to SEQ ID NO. 9.

16. The method according to claim 11, wherein the polynucleotide encoding
DAP comprises a sequence encoding a protein a having at least about 85
percent homology to SEQ ID NO. 9.

17. The method according to claim 11, wherein the subject is a mammal.

18. The method according to claim 11, wherein the subject is a human.

Description:

[0002] Type 1 diabetes, often referred to as juvenile diabetes, is
associated with islet cell dysfunction which results in loss of insulin
production. Understanding how hormones such as insulin and glucagon are
produced and regulated for secretion in islets of the pancreas is
important for the prevention and treatment of type 1 diabetes.

[0006] PTPRN was recently found in kidney as well. Knockout of the Ptprn
gene in mice resulted in a marked reduction of renin-angiotensin levels
in plasma (S M Kim et al., Am. J. Physiol. Renal Physiol., 296, F382-389
(2009)), which suggests that DAP and PTPRN play a similar role in
regulating blood pressure in kidney. The RESP18 protein was also found in
the kidney tissue by Western blots (M R Schiller et al., J. Biol. Chem.,
270, 26129-26138 (1995)). However, whether DAP is colocalized with PTPRN
and/or RESP18 in the same cells of kidney needs further investigation.

[0007] The major renin-angiotensin system (RAS) components, including
angiotensinogen, Angiotensin I (Ang I), Angiotensin II (Ang II),
Angiotensin III (Ang III), Renin, and Angiotensin-Converting Enzyme
(ACE), were found in islets and islet MIN6 cells. Ramracheya et al.,
Diabetologia, 49, 321-331 (2006); Leung et al., Adv. Exp. Med. Biol.,
654, 339-361 (2010). The local RAS plays paracrine and autocrine roles in
the regulation of various functions, such as cell proliferation,
apoptosis, and hormone secretion, in islets. Angiotensinogen is only
expressed in alpha cells and not in other islet cells. Regoli et al., J.
Endocrinol., 179, 81-89 (2003). Ang II, known for its blood pressure
maintenance and body fluid balance functions, has been implicated in the
regulation of the local islet RAS. Beyond the role in the regulation of
blood pressure, Ang II can also regulate insulin signaling, thus leading
to insulin resistance and diabetes.

SUMMARY

[0008] The methods provided herein are based on the discovery, as
described in more detail herein, that aspartyl aminopeptidase (DAP) is
expressed in mammalian pancreatic islet cells and that increased DAP
expression results in decreased Angiotensin II (Ang II) levels in the
islet cells. Conversely, it was also found that inhibition of DAP
expression resulted in increased Ang II levels. It has not been
previously reported that DAP was expressed in mammalian pancreatic islet
cells or that DAP expression levels in the islet cells impacted
intracellular or extracellular Ang II levels.

[0009] By one approach, a screening method is provided for identifying a
compound effective to increase DAP activity in mammalian pancreatic islet
cells, the method comprising: contacting purified DAP and Ang II with a
test compound; measuring the level of Ang II; and determining if the
level of Ang II is lower when treated with the test compound than an
otherwise identical control containing DAP and Ang II that has not been
contacted with the test compound. In one aspect, DAP and Ang II are
contacted with the test compound for an amount of time effective for DAP
to degrade at least about 10 percent of the Ang II present so that
differences in activity between the test sample and the control sample
can be detected. By this approach, compounds that are effective cofactors
or enzyme activators can be identified.

[0010] In another aspect, a screening method is provided for identifying a
compound that restrains, blocks, or suppresses a DAP inhibitor and
thereby increases DAP activity in mammalian pancreatic islet cells upon
inactivation of the inhibitor. The method comprises contacting an islet
cell lysate with a test compound and purified DAP and Ang II; measuring
the level of Ang II; and determining if the level of Ang II is lower when
treated with the test compound than an otherwise identical control
containing the cell lysate, DAP, and Ang II that has not been contacted
with the test compound.

[0011] Once it has been determined that a DAP inhibitor is present in the
lysate, a screening method is provided for identifying a compound
effective to increase DAP expression in mammalian pancreatic islet cells,
the method comprising: culturing mammalian pancreatic islet cells capable
of producing DAP in the presence of a test compound; measuring the level
of DNPEP gene expression; determining if the level of DNPEP gene
expression is higher when treated with the test compound than an
otherwise identical control not cultured with the test compound. The
level of gene expression can be determined by measuring the amount of DAP
and/or the amount of mRNA that encodes DAP. By another approach, the
method may further comprise measuring the level of Ang II and determining
if the level of Ang II is lower when treated with the test compound.

[0012] The disclosure also provides a method for decreasing Ang II levels
in a subject having Ang II induced pancreas islet cell dysfunction and/or
suffering or susceptible to type 1 diabetes, the method comprising
administering to the subject a therapeutically effective amount of a test
compound identified according to the methods described herein, where the
test compound is effective to increase the expression or activity of DAP
in mammalian pancreatic islet cells. In another aspect, a method is
provided for the treatment or prevention of type 1 diabetes comprising
administering to the subject a therapeutically effective amount of a test
compound identified according to the methods herein. It is also presently
believed that reducing the Ang II content of the pancreatic islet cells
may be effective at controlling insulin resistance and, hence, type II
diabetes.

[0013] In yet another aspect, a method is provided for increasing DAP
expression and decreasing Ang II levels in a subject, the method
comprising delivering one or more copies of a polynucleotide encoding DAP
to the subject's pancreatic islet cells. By one approach, the one or more
copies of a polynucleotide encoding DAP can be delivered via a viral
vector.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The foregoing features, as well as other features, will become
apparent with reference to the description and Figures below.

[0015] FIG. 1 is a schematic showing DAP and the islet renin-angiotensin
system (RAS) metabolism in islet cells. The major RAS components,
including angiotensinogen, Ang I, Ang II, Ang III, Renin, and ACE, were
found in islets and MIN6 cells. ("+" indicates activation or increase;
"-" indicates inhibition or decrease.)

[0016] FIGS. 2A-E are microscope images taken with a Zeiss Axiophot
microscope, which illustrates how PTPRN (FIG. 2A), RESP18 (FIG. 2B); DAP
(FIG. 2C), and glucagon (FIG. 2D) are expressed in islet cells. All
images are presented in 20× amplification. PTPRN (FIG. 2A) and are
expressed in pancreatic islet cells with stronger signal in peripheral
alpha cells. DAP (FIG. 2C) has a similar expression pattern to that of
glucagon-positive cells (FIG. 2D), and is completely overlapped with the
glucagon signal (FIG. 2E), as determined by double stainings with
antibodies to DAP and glucagon separately.

[0017] FIG. 3 is representative images of in situ hybridization in the
adult mouse brain (sagittal). Scale bars are indicated in microns. mRNAs
of Dnpep, Resp18 and Ptprn are detected with a similar pattern in various
brain regions, including midbrain (mb), medulla (my), pons (p), thalamus
(th), subthalamic nucleus (sn), hippocampal region (hip), main olfactory
bulb (mob), cerebral cortex (ctx), caudate putamen (cp), and cerebellum
(ctx) as judged by staining in a sagittal section of mouse brain from the
Allen Brain Atlas.

[0018] FIG. 4 are representative in situ expression images of Dnpep,
Resp18 and Ptprn mRNAs in cervical spinal cord of adult mice. Scale bars
are indicated in microns. All of the three genes are mainly expressed in
gray matter that is shaped like a butterfly which contains interneurons
and motor neurons.

[0022] The methods provided herein are based on the discovery, as
described in more detail herein, that aspartyl aminopeptidase (DAP) is
expressed in mammalian pancreatic islet cells and that increased DAP
expression results in decreased Ang II levels in the islet cells.
Conversely, it was also found that inhibition of DAP expression resulted
in increased Ang II levels. It was not previously reported that DAP was
expressed in mammalian pancreatic islet cells or that DAP expression
levels in the islet cells impacted intracellular and extracellular Ang II
levels.

[0023] Many homologous genes are clustered in genomes and evolutionarily
conserved in syntenic segments cross-species. DNPEP is an evolutionarily
conserved gene found from C. elegans to human. It was discovered that
DNPEP was physically linked with PTPRN in genomes in both human and C.
elegans, suggesting a strong syntenic conservation of the block over
evolutionary spectrum of bilaterian animals. Conserved genes within the
same syntenic block may keep similar tissue specific expression patterns
in neuroendocrine tissues including islets and neuronal tissues in the
brain and spinal cord and hence may also be involved in similar
biological functions.

[0024] Through genomic localization, tissue specific expression,
subcellular localization and enzyme activity studies presented herein, it
was demonstrated that DAP and Protein Tyrosine Phosphatase Receptor type
N (PTPRN) are colocalized in the secretory granules of pancreatic islet
cells. It was also found that DAP was colocalized with glucagon in
hormone secretory granules in pancreatic islet cells. In situ
hybridization analysis showed very similar expression patterns of Dnpep,
Resp18 and Ptprn in both the brain and spinal cord. Since genes expressed
in the same tissue and cellular localization may have similar regulatory
elements in their highly conserved noncoding elements (HCNEs),
identification of such genomic regulatory blocks (GRBs) with specific
expression patterns in pancreatic endocrine cells may provide clues to
promote further investigation in corresponding biological functions of
these genes. These findings suggest that genes from this syntenic block
may also function coordinately. This colocalization signifies that DAP
may play an important role in posttranslational processing and secretion
of hormones.

[0025] Since the 5'-terminal flanking region of DNPEP showed considerable
similarity to that of PTPRN and RESP18, it appeared that the expression
of these genes may be regulated by common transcription factor(s) for
their specific expression in islet cells. It was also found that DAP was
expressed in mouse and human islet alpha cells and was subcellularly
associated with secretory granules and lysosomal-like compartments. DAP
enzymatic activity assay demonstrated that the highest activities of DAP
was detected in synaptosomal- and lysosomal-enriched cellular fractions
of brain tissues. These findings support the notion that genes located in
the same syntenic block may also share similar expression patterns and
even related biological functions.

[0026] As described further in the Examples herein, it was found that
upregulated DAP expression decreases the intracellular and extracellular
Ang II levels by deleting the N-terminal aspartic (Asp) of both Ang I
(1-14) (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser; SEQ ID
NO. 6) and Ang II (1-8) (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe; SEQ ID NO. 7),
which provides Ang I* (des-Asp-Ang I) and Ang III (des-Asp-Ang II),
respectively. Ang I appears to have no biologically activity and exists
solely as a precursor to Ang II. Ang II can regulate insulin signaling,
thus leading to insulin resistance and diabetes through a complicated
process as shown in FIG. 1. After being processed by renin and
angiotensin I converting enzyme (ACE), Ang II is secreted and binds to
the type-1 angiotensin receptor (AT1) on beta cells, which activates
downstream JAK2-STAT pathway. In pathological conditions, over-activation
of Ang II and AT1 receptor results in islet beta cell dysfunction and
diabetes. Accordingly, DAP can be targeted by potential small molecules
to prevent or control type 1 diabetes.

[0027] The discovery of the relationship between the expression of DAP,
the gene product of DNPEP, and Ang II levels in mammalian pancreatic
islet cells can be utilized to identify compounds capable of modulating
DAP levels in the islet cells. In one aspect, a method is provided for
identifying a compound that increases the expression of DAP, the gene
product of DNPEP, in mammalian pancreatic islet cells. In another aspect,
a method is provided for identifying a compound that increases DAP
activity in mammalian pancreatic islet cells. In both aspects, the
increased DAP expression or activity results in decreased angiotensin II
(Ang II) levels in the mammalian pancreatic islet cells, thereby
preventing Ang II induced pancreas islet cell dysfunction and preventing
or reversing diabetes.

[0028] In another aspect, a method can then be provided for treating or
preventing type 1 diabetes by administering a pharmaceutical composition
comprising an effective amount of the identified compound which is
effective to increase the expression or activity of DAP in mammalian
pancreatic islet cells and thereby decreasing Ang II levels.

[0029] Terms

[0030] The term "compound" is used in the context of a "test compound" or
a "drug candidate compound" described in connection with the assays
described herein. Test compounds include, for example, peptides,
proteins, antibodies, non-peptide compounds, synthetic compounds,
fermentation products, cell extracts, plant extracts, animal tissue
extracts, plasma and the like. As such, these compounds comprise organic
or inorganic compounds, derived synthetically or from natural sources. In
one aspect, libraries of compounds can be used for high-throughput
purposes. Suitable libraries include, for example, antibody fragment
libraries, lipid libraries, synthetic compound libraries, natural
compound libraries, and peptide libraries. Other libraries may be used,
if desired. The compounds also include pharmaceutically acceptable salts
thereof.

[0031] The term "contact" or "contacting" means bringing at least two
moieties together, including both in vitro and in vivo systems.

[0032] The term "condition" or "disease" means the presentation of
symptoms (i.e., illness) or the manifestation of abnormal clinical
indicators (e.g., biochemical indicators), resulting from islet cell
dysfunction, including type I diabetes. Alternatively, the term "disease"
refers to a genetic or environmental risk of or propensity for developing
such symptoms or abnormal clinical indicators.

[0033] The term "effective amount" or "therapeutically effective amount"
means that amount of a compound that will elicit the biological or
medical response of a subject that is being sought by a medical doctor or
other clinician. In one aspect, particularly with regard to treating type
1 diabetes, the term "effective amount" is intended to mean the amount of
a compound or agent that will bring about a biologically meaningful
decrease in the levels of Ang II in the subject's islet cells.

[0034] The term "expression" comprises both endogenous expression and
overexpression by transfection.

[0035] The terms "substantially pure" or "substantially purified" as used
herein in reference to a given polypeptide means that the polypeptide is
substantially free from other biological macromolecules. For example, the
substantially pure polypeptide is at least about 70 percent, in another
aspect at least about 75 percent, in another aspect at least 80 percent,
in another aspect at least about 85 percent, in another aspect at least
about 90 percent, and in yet another aspect at least about 95 percent
pure by dry weight. Purity can be measured by any appropriate standard
method known in the art, such as, but not limited to, column
chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. For
use in these experiments, the polypeptides used herein, such as DAP
and/or Ang II, can be purified using known techniques, including, for
example, salt precipitation, differential solubilization, chromatography
(e.g., high performance liquid chromatography, ion exchange
chromatography, gel permeation chromatography, hydrophobic interaction
chromatography, affinity chromatography, and immunoaffinity
chromatography), centrifugation, and combinations thereof.

[0036] The term "subject" includes mammals and specifically includes
humans.

[0037] The term "treating" means an intervention performed with the
intention of preventing the development or altering the pathology of, and
thereby alleviating a disorder, disease or condition, including one or
more symptoms of such disorder or condition. Accordingly, "treating"
refers to both therapeutic treatment and prophylactic or preventative
measures. The related term "treatment," as used herein, refers to the act
of treating a disorder, symptom, disease or condition, as the term
"treating" is defined above. Those in need of treatment include those
already with the disorder as well as those in which the disorder is to be
prevented.

[0038] The term "Angiotensin II" or "Ang II" as used herein includes a
protein having the amino acid sequence of SEQ ID NO: 7, or a polypeptide
in which one or more amino acids is modified by deletion, addition,
and/or substitution by another amino acid, wherein said protein is
functionally equivalent to the polypeptide having the amino acid sequence
of SEQ ID NO: 7.

[0039] The nucleotide sequence of the DAP cDNA and the amino acid sequence
of the protein encoded by the cDNA are shown in SEQ ID NOs: 8 and 9,
respectively. The term "aspartyl aminopeptidase" or "DAP" as used herein
includes a protein having the amino acid sequence of SEQ ID NO: 9, or a
protein in which one or more amino acids is modified by deletion,
addition, and/or substitution by another amino acid, wherein said protein
is functionally equivalent to the protein having the amino acid sequence
of SEQ ID NO: 9. In one aspect, the protein has at least 80 percent
identity to the amino sequence of SEQ ID NO: 9, in another aspect at
least 85 percent identity, in another aspect at least 90 percent
identity, and in yet another aspect at least 95 percent identity. It is
known in the art that a protein may have an amino acid sequence which is
modified by deletion, addition, and/or substitution by other amino acids
yet still retain its biological activity.

[0040] The hybridization technique (Sambrook et al., Molecular Cloning 2nd
ed. 9.47-9.58, Cold Spring Harbor Lab. press, 1989) is well known to
those of skill in the art as an alternative to preparing a protein
functionally equivalent to a certain protein. More specifically, one
skilled in the art can utilize the general procedure to obtain a protein
functionally equivalent to human DAP by isolating DNA having a high
homology with the whole or part of the cDNA (SEQ ID NO: 8) encoding human
DAP. Accordingly, the term "DAP" as used herein includes such proteins
that are encoded by DNA that hybridizes with cDNA encoding human DAP or
part thereof and that are functionally equivalent to a human DAP. For
instance, homologues of human DAP in other mammals (such as those of
monkey, mouse, rabbit, and bovine) are included.

[0041] Stringent hybridization conditions for isolating DNA encoding
functionally equivalent proteins of human DAP can be suitably selected by
one skilled in the art, and for example, low-stringent conditions can be
given. Low-stringent conditions are, for example, 42° C.,
2×SSC, and 0.1% SDS, and in another aspect, 50° C.,
2×SSC, and 0.1% SDS. In some aspects, highly stringent conditions
are more preferable and include, for example, 65° C., 2×SSC,
and 0.1% SDS. Under these conditions, the higher the temperature, the
higher the homology of the obtained DNA. However, several factors other
than temperature, such as salt concentration, can influence the
stringency of hybridization and one skilled in the art can suitably
select the factors to accomplish a similar stringency.

[0042] Alternatively, a gene amplification method, for example, the
polymerase chain reaction (PCR) method, can be utilized to isolate the
object DNA using primers synthesized based on the sequence information of
the DNA encoding human DAP (SEQ ID NO: 8). One of ordinary skill in the
art of design of amplification primers will recognize that a given primer
need not hybridize with 100 percent complementarity in order to
effectively prime the synthesis of a complementary nucleic acid strand in
an amplification reaction. Moreover, a primer may hybridize over one or
more segments such that intervening or adjacent segments are not involved
in the hybridization event (e.g., a loop structure or a hairpin
structure). In one aspect, the primers are at least 13 nucleotides in
length. In another aspect, the primers are less than 36 nucleotides in
length. In some aspects, the function of a given primer may be
substituted by a combination of two or more primers that hybridize
adjacent each other or that are linked by a nucleic acid loop structure
or linker which allows a polymerase to extend the two or more primers in
an amplification reaction.

[0044] By one approach, methods are provided for identifying a compound
which is effective to increase the activity of DAP, either directly or
indirectly, in mammalian pancreatic islet cells. By "increased activity"
is meant that the amount of Ang II degraded by DAP per unit of time is
higher in the presence of the compound while the amount of DAP,
temperature, pH, and other environmental variables are held constant. In
one aspect, activity is increased by at least about 10 percent, in
another aspect at least about 20 percent, in another aspect at least
about 30 percent, in another aspect at least about 40 percent, in another
aspect at least about 50 percent, in another aspect at least about 60
percent, and in yet another aspect at least about 75 percent. A compound
may be effective to increase DAP activity by several mechanisms as
discussed below.

[0045] In one aspect, the identified compound may be a cofactor that is
necessary for enzymatic activity of DAP. Cofactors may include any
non-protein compound, such as, for example, organic molecules, such as
coenzymes and prosthetic groups, or inorganic molecules, such as metallic
cations. In some aspects, at least one cofactor may be needed for the
enzymatic activity of DAP. While the cofactor may be found in the
pancreatic islet cells, the cofactor may be present in an insufficient
quantity such that the amount present in the cells limits DAP activity.
For example, the cofactor may be present in limited quantity in diseased
cells. Therefore, providing increased amounts of cofactor to the islet
cells is expected to be effective to increase DAP activity.

[0046] In another aspect, the identified compound may be an enzyme
activator that is effective to increase DAP activity. An activator is a
substance other than a substrate or cofactor that is effective to
increase the rate of the enzymatic reaction.

[0047] In another aspect, the identified compound may be effective to
increase DAP activity indirectly by interfering with an inhibitor of DAP
activity. For example, the compound may interfere by restraining,
blocking, or suppressing the DAP inhibitor. An enzyme inhibitor is a
molecule that decreases the enzyme's activity after binding to the
enzyme. The inhibitor's interaction with the enzyme can be reversible or
irreversible. There may be one or more inhibitors of DAP in the islet
cells which are involved in the regulation of DAP activity in the cells.
For example, the inhibitor may be involved in a negative feedback loop
where accumulation of a downstream product inhibits future production by
the system. Such negative feedback mechanisms are known to be involved in
the regulation of many types of hormones. In some cases, the inhibitor
may bind to DAP in its active site or may bind to another portion of DAP,
such as a regulatory site, to reduce DAP's activity or affinity for
substrate. In some aspects, the inhibitor may reversibly bind to DAP by
non-covalent interactions, such as hydrogen bonding, ionic bonding, or
hydrophobic interaction. Reversible inhibitors may act by competitive
inhibition, uncompetitive inhibition, non-competitive inhibition, or
mixed inhibition.

[0048] In other aspects, the inhibitor may be an irreversible inhibitor
having a reactive functional group that covalently modifies DAP by
reacting with amino acids of DAP to form covalent adducts. Because the
irreversible inhibitor binds to the enzyme, the covalent adduct has a
greater mass than unmodified DAP. The presence of an irreversible
inhibitor can be identified by mass spectrometry or by using DAP-specific
antibody in Western blot analysis.

[0049] A compound can be differentiated as being effective to increase DAP
activity as opposed to being effective to increase DAP expression by
several techniques.

[0050] In one aspect, the assay includes a cell free assay system that is
not capable of gene expression. By one approach, a method is provided for
identifying a compound effective to increase DAP activity in mammalian
pancreatic islet cells, the method comprising: contacting DAP and Ang II
with a test compound; measuring the level of Ang II; and determining if
the level of Ang II is lower when treated with the test compound than an
otherwise identical control containing DAP and Ang II that has not been
contacted with the test compound. By one approach, the contacting step is
carried out at conditions similar to physiological conditions in
pancreatic islet cells. In one aspect, the contacting step is carried out
in an aqueous solution at a temperature of about 32° to about
40° C., in another aspect about 35° C. to about 39°
C., and in another aspect at a temperature of about 37° C. and at
a pH of about 7.2 to about 7.6, in another aspect at about 7.4. By one
approach, the aqueous solution is phosphate buffered saline (PBS). At
least in some approaches, the DAP and/or Ang II used is in a
substantially purified form.

[0051] In one aspect, DAP and Ang II are contacted with the test compound
for an amount of time effective for DAP to degrade at least about 10
percent of the Ang II present so that differences in activity between the
test sample and the control sample can be detected. For example, the DAP
and Ang II is contacted with the test compound for at least about 30
minutes, in another aspect at least about 45 minutes, and in another
aspect at least about 60 minutes, or any other amount of time effective
for a difference in activity to be observed between a control and test
sample. By this approach, compounds that are effective cofactors or
enzyme activators can be identified.

[0052] In another aspect, an islet cell lysate can be prepared and
contacted with purified DAP and Ang II to determine if there is a
compound present in the cell lysate that acts to decrease or inhibit DAP
activity compared to an otherwise identical control that does not include
cell lysate. Suitable islet cells include cells capable of producing DAP
and Ang II. For example, MIN6 cells, which is a cell line derived from in
vivo immortalized insulin-secreting pancreatic beta cells, can be used.
The cell lysate can be prepared after culturing islet cells to suitable
confluence, such as about 80 percent confluence, washing the cells with
PBS, and lysing the cells with a freeze/thaw process, such as, for
example, by freezing at -20° C., -80° C., or in dry
ice/ethanol bath and thawing in a room temperature or 37° C. water
bath. Generally, more than one freeze/thaw cycles may be necessary for
efficient lysis of the cells. Other lysis techniques may be used, if
desired, including homogenization, sonication, or chemical lysis, but the
lysis technique selected should not adversely impact the enzymatic
activity of DAP. The recovered lysate should be incapable of protein
expression. Samples are then prepared containing purified DAP and Ang II
which are then contacted with and without the lysate to determine if the
lysate contains an inhibitor of DAP activity.

[0053] If an inhibitor is present, the sample contacted with the lysate
will contain higher levels of Ang II after a period of incubation than
the sample not containing the lysate. In one aspect, the period of
incubation may be at least about 30 minutes, in another aspect at least
about 45 minutes, and in another aspect at least about 60 minutes, or any
other amount of time effective for a difference in activity to be
observed between a control and test sample.

[0054] If it is found that there is an inhibitor of DAP activity in the
cell lysate, a screening method is provided for identifying a compound
that interferes with the DAP inhibitor and thereby increases DAP activity
in the mammalian pancreatic islet cells. The method comprises contacting
an islet cell lysate with a test compound and DAP and Ang II; measuring
the level of Ang II; and determining if the level of Ang II is lower when
treated with the test compound than an otherwise identical control (i.e.,
containing the cell lysate, DAP, and Ang II) that has not been contacted
with the test compound. At least in some approaches, the DAP and/or Ang
II used is in a substantially purified form.

[0055] At least in some approaches, the cell lysate is treated to separate
protein from smaller molecules to further characterize the inhibitor. A
variety of techniques can be used. For example, a density gradient
fractionalization system, which separates by size, may be used to
identify and/or isolate cell lysate components of interest. Membrane
filtration may also be used, if desired, with a membrane of suitable
molecular weight cutoff.

[0056] Protein levels can be measured by conventional levels. By one
exemplary approach, the islet cells can be grown in DMEM, supplemented
with 10 percent fetal calf serum (FCS), 10 mM glucose, and 2 mmol/l
glutamine, and are grown to suitable confluence, such as at least about
80 percent confluence, prior to harvesting. The cells are generally grown
at a temperature of about 35° C. to about 39° C., in
another aspect about 37° C. The cell lysate can be prepared after
culturing islet cells to about 80 percent confluence, washing the cells,
such as with PBS, and lysing the cells with a freeze/thaw process as
previously described. The levels of DAP and Ang II levels can then be
detected by conventional techniques, such as Western blot analysis,
ELISA, or a method based thereon using an antibody that recognizes DAP
and/or Ang II. Other conventional methods of detecting protein levels may
be used, if desired.

[0057] Ang II levels can be detected by conventional techniques. By one
approach, Ang II levels can be measured using the AssayMax Ang II ELISA
Kit (Cayman Chemical, Ann Arbor, Mich.). By this approach, standards or
samples can be incubated in a 96-well plate with biotinylated anti-Ang II
antibody for 2 hours. After the washings, Streptavidin-Peroxidase
Conjugate is added for 30 minutes. The final reaction is developed with
the substrate chromogen and read at 450 nm by an ELISA reader. Ang II
levels in the samples can be calculated from an Ang II standard curve run
with each assay.

[0058] At least in some approaches, a test compound that increases the
activity of DAP by at least about 20 percent, in another aspect at least
about 30 percent, in another aspect at least about 30 percent, in another
aspect at least about 40 percent, in another aspect at least about 50
percent, in another aspect at least about 60 percent, and in yet another
aspect at least about 75 percent, can be selected as a compound that
increases DAP activity and decreases Ang II levels in mammalian
pancreatic islet cells.

[0059] By another approach, methods are provided for identifying compounds
which are effective to increase the expression of DAP in mammalian
pancreatic islet cells. By "increased expression" is meant that the rate
of gene transcription is increased or mRNA stability is increased such
that the amount of DAP or the amount of mRNA that encodes DAP produced is
measurably higher when pancreatic islet cells are treated with the test
compound than without the test compound. In one aspect, expression is
increased by at least about 10 percent, in another aspect at least about
20 percent, in another aspect at least about 30 percent, in another
aspect at least about 40 percent, and in yet another aspect at least
about 50 percent.

[0060] In one aspect, the compound may effect increased expression of DAP
by acting as a transcription factor. For example, the compound may
promote recruitment or stabilization of RNA polymerase to the DNPEP gene.
In another aspect, the compound may act as a transcription factor by
recruiting a coactivator to the DNPEP gene. A transcription factor
generally includes one or more DNA-domains for attachment to portions of
the DNA adjacent the gene to be regulated. The transcription factor may
bind to either the enhancer or promoter region of the DNPEP gene. In yet
another aspect, the compound may act as a coactivator (a substance that
works with one or more transcription factors) to increase the rate of
transcription from the DNPEP gene.

[0061] In another aspect, the compound may effect increased expression of
DAP by binding to a repressor or corepressor of gene transcription from
the DNPEP gene. Repressors generally act by attaching to the operator of
the gene and prevent RNA polymerase from attaching to the gene and
transcribing the gene to produce mRNA. A corepressor acts by binding to
the repressor so that the repressor binds more tightly to the operator.
In one aspect, the compound may act to increase DAP expression by binding
to the operator portion of the DNPEP gene, thereby preventing binding of
a repressor. In another aspect, the compound may bind to the repressor,
thereby preventing binding of the repressor to the gene. Providing such
compounds to islet cells is expected to result in increased DAP
expression.

[0062] In one aspect, a screening method is provided for identifying a
compound effective to increase DAP expression in mammalian pancreatic
islet cells, the method comprising: culturing mammalian islet cells
capable of producing DAP in the presence of a test compound; measuring
the level of DNPEP gene expression; and determining if the level of DNPEP
gene expression is higher when treated with the test compound than an
otherwise identical control not cultured with the test compound.

[0063] In another aspect, a screening method is provided for identifying a
compound effective to increase DAP expression in mammalian pancreatic
islet cells, the method comprising: culturing mammalian islet cells
capable of producing DAP in the presence of a test compound; measuring
the level of DNPEP gene expression; and determining if the level of DNPEP
gene expression is higher when treated with the test compound than an
otherwise identical control not cultured with the test compound. The
level of gene expression can be determined by measuring the amount of DAP
and/or the amount of mRNA that encodes DAP. By another approach, the
method may further comprise measuring the level of Ang II and determining
if the level of Ang II is lower when treated with the test compound.

[0064] In one aspect, the mammalian islet cells are contacted with the
test compound for an amount of time sufficient to detect increased
transcription from DNPEP. In another aspect, the mammalian islet cells
are contacted with the test compound for an amount of time sufficient to
detect increased transcription from DNPEP and for any DAP produced to
degrade at least about 10 percent of the Ang II present so that
differences in expression levels between the test sample and the control
sample can be detected. For example, the cells are contacted with the
test compound for at least about 30 minutes and in another aspect at
least about 60 minutes.

[0065] Any mammalian islet cells capable of producing DAP and Ang II can
be used in the methods described herein. For example, suitable islet
cells include, for example, MIN6 cells, which is a cell line derived from
in vivo immortalized insulin-secreting pancreatic beta cells.

[0066] Protein levels can be measured by conventional methods. By one
approach, the islet cells can be grown in DMEM, supplemented with 10%
FCS, 10 mM glucose, and 2 mmol/l glutamine, and are grown to and are
grown to suitable confluence, such as at least about 80 percent
confluence, prior to harvesting. The cells are generally grown at a
temperature of about 35° C. to about 39° C., in another
aspect about 37° C. The cells can then be washed, such as with
PBS, and lysed, such as by freeze/thaw as described above. The levels of
DAP and Ang II levels in the lysate can then be detected by conventional
techniques, such as Western blot analysis, ELISA, or a method based
thereon using an antibody that recognizes DAP. Other conventional methods
of detecting protein levels may be used, if desired. By one approach, Ang
II levels can be detected by conventional techniques.

[0067] By one approach, Ang II levels can be measured using the AssayMax
Ang II ELISA Kit (Cayman Chemical, Ann Arbor, Mich.). By this approach,
standards or samples can be incubated in a 96-well plate with
biotinylated anti-Ang II antibody for 2 hours. After the washings,
Streptavidin-Peroxidase Conjugate is added for 30 minutes. The final
reaction is developed with the substrate chromogen and read at 450 nm by
an ELISA reader. Ang II levels in the samples can be calculated from an
Ang II standard curve run with each assay.

[0068] The amount of mRNA can be measured according to conventional
methods. The mRNA is isolated from the islet cells. For example, Northern
blot analysis using as the probe a nucleic acid capable of binding to the
mRNA, or Reverse Transcription PCR using as the primer a nucleic acid
capable of hybridizing to the mRNA a portion thereof. Other conventional
methods of detecting mRNA levels may also be used, if desired.

[0069] By one approach, small interfering RNA (siRNA) can be used to
confirm that the test compound is effective for increasing DAP
expression, rather than increasing DAP activity. By this approach, siRNA
is provided that is sufficiently homologous to a portion of mRNA encoding
DAP such that the siRNA interferes with the translation of the DAP mRNA.
The siRNA can be transfected into the cells using an appropriate vector,
such as pCMV-SPORT6 (Open Biosystems, Huntsville, Ala.) by
electroporation (BioRad Laboratories, Berkeley, Calif.). By use of the
siRNA, the endogenous DAP mRNA level and DAP protein level should be
substantially reduced while Ang II levels should increase.

[0070] At least in some approaches, a test compound that increases the
expression level of DAP by at least about 20 percent, in another aspect
at least about 30 percent, in another aspect at least about 30 percent,
in another aspect at least about 40 percent, in another aspect at least
about 50 percent, in another aspect at least about 60 percent, and in
another aspect at least about 75 percent, can be selected as a compound
that promotes the expression of DAP or the expression of the DNPEP gene
for the protein and decreases Ang II levels in mammalian pancreatic islet
cells.

Delivery of One or More Additional Copies of DNPEP to Increase DAP
Production

[0071] By another approach, one or more additional copies of the DNPEP
gene can be delivered to mammalian pancreatic islet cells for increasing
in vivo expression of DAP and thereby decreasing levels of Ang II in the
cells. A variety of viral-based delivery systems, including adenoviral,
retroviral, adeno-associated viral, lentiviral, and herpes simplex viral
systems, can be used to introduce one or more additional copies of the
DNPEP gene in target cells.

[0072] In one aspect, a polynucleotide encoding DAP is incorporated into a
viral vector. It is possible to use such a construct to perform gene
therapy for diseases that arise from mutations in the DNPEP gene.
Conventional techniques can be used to insert a polynucleotide encoding
DAP (e.g., cDNA of SEQ ID NO. 8) into the viral vector. In one aspect,
the polynucleotide comprises a sequence encoding a protein a having at
least about 80 percent homology, in another aspect at least about 85
percent, in another aspect at least about 90 percent, and in yet another
aspect at least about 95 percent homology to SEQ ID NO. 9.

[0073] In some aspects, in the construction of the vector, the
polynucleotide encoding DAP may be linked to one or more regulatory
regions, including promoters, enhancers, suppressors, and the like.
Promoters that may be used include both constitutive and regulated (e.g.,
inducible) promoters. By one approach, a cell specific promoter is used.
For example, a promoter may be used so that the gene is only expressed in
pancreatic alpha cells. For example, a glucagon-activated promoter can be
used. A suitable glucagon-activated promoter is provided at SEQ ID NO.
10.

[0074] In some aspects, the viral vectors used herein lack at least one
region necessary for replication of the virus in the target cell and are
replication defective.

[0075] The vector may then be administered to the subject through ex vivo
or in vivo methods. By this approach, the levels of DAP in the target
cells can be increased. The increased DAP levels increase the degradation
of Ang II, thereby reducing Ang II levels in the cells and preventing Ang
II induced pancreas islet cell dysfunction.

Pharmaceutical Compositions

[0076] Therapeutic methods and pharmaceutical compositions which include
effective amounts of the test compounds or pharmaceutically acceptable
salt thereof identified herein are also provided. In some aspects, the
compositions may be useful for treating or preventing type 1 diabetes.

[0077] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in the
art in dosages suitable for oral administration. Appropriate carriers can
be selected to formulate the compositions in the form of, for example,
tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for ingestion by the subject. Excipients can
also be included, such as carbohydrate or protein fillers, such as
sugars, including lactose, sucrose, mannitol, or sorbitol; starch from
corn, wheat, rice, potato, or other plants; cellulose, such as
methylcellulose, hydroxypropylmethyl-cellulose, or sodium
carboxymethyl-cellulose; gums including arabic and tragacanth; and
proteins such as gelatin and collagen. If desired, the pharmaceutical
composition may be prepared in delayed release, sustained release, or
other format. The composition may also include an enteric coating, if
desired.

[0078] Injectable formulations can also be prepared, such as, for example,
in the form of a solution or suspension in a non-toxic,
parenterally-acceptable solvent or diluent. Exemplary
pharmaceutically-acceptable carriers include saline, buffered saline,
isotonic saline, Ringer's solution, dextrose, water, sterile water,
glycerol, ethanol, and combinations thereof.

[0079] The disclosure also provides a method of decreasing Ang II levels
in a subject having Ang II induced pancreas islet cell dysfunction, the
method comprising administering to the subject a therapeutically
effective amount of a test compound identified according to the methods
described herein, where the test compound is effective to increase the
expression or activity of DAP in mammalian pancreatic islet cells. In one
aspect, the subject may have or be susceptible to type 1 diabetes. In
another aspect, a method is provided for the treatment or prevention of
type 1 diabetes comprising administering to the subject a therapeutically
effective amount of a test compound identified according to the methods
herein.

[0080] As it is known that Ang II can cause insulin resistance, it is also
presently believed that reducing the Ang II content of the pancreatic
islet cells may be effective to at least partially and possibly
significantly reducing insulin resistance and thereby controlling type II
diabetes.

[0081] As used herein, therapeutically effective amount or dose means the
amount of compound which ameliorates the symptoms or condition.
Therapeutic efficacy and toxicity of such compounds can be determined by
standard pharmaceutical procedures in cell cultures or experimental
animals, e.g., ED50 (the dose therapeutically effective in 50% of the
population) and LD50 (the dose lethal to 50% of the population). The dose
ratio of toxic to therapeutic effects is the therapeutic index, and it
can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions
that exhibit large therapeutic indices are preferred. Data obtained from
cell culture assays and animal studies can be used in formulating a range
of dosage for human use. The dosage may vary depending upon the dosage
form employed, sensitivity of the patient, and the route of
administration. The dosage suitable for a given subject can be readily
determined by one of skill in the art. Generally, dosage and
administration can be adjusted to provide sufficient levels of the test
compound identified herein or to maintain the desired effect.

[0082] The pharmaceutical compositions provided herein may be administered
to a subject by a variety of methods. In one aspect, the compositions may
be applied directly to target tissues, such as the pancreas. In one
aspect, local administration to the desired tissue may be done by
catheter, infusion pump or stent. Additional routes of delivery include,
for example, intravenous injection, intramuscular injection, subcutaneous
injection, aerosol inhalation, oral (tablet or pill form), topical,
systemic, ocular, intraperitoneal and/or intrathecal delivery.

[0083] In the case of delivery of recombinant viruses as discussed above,
recombinant viruses are formulated and administered in the form of doses
of an appropriate number of plaque forming units (pfu). The term pfu
corresponds to the infective power of a suspension of virions and is
determined by infecting an appropriate cell culture and measuring the
number of plaques formed. The techniques for determining the pfu titre of
a viral solution are well documented in the prior art and the desired
dose of recombinant virus can be readily determined by one of ordinary
skill in the art.

[0084] The following Examples are provided to illustrate certain
embodiments of the invention but should not be construed as limiting the
scope of the disclosure. All publications and patents listed in this
disclosure are incorporated herein in their entirety.

EXAMPLES

Example 1

[0085] Bioinformatic Analysis

[0086] The entire sequence and predicted genes of human DNPEP-containing
BAC clone (GenBank acc. no. AC053503) were analyzed by BLAST searching
the National Center for Biotechnology Information databases with BLAST
algorithms. The adjacent genes of DNPEP including PTPRN and RESP18 were
investigated to determine whether the orthologues could be mapped to the
vicinity of DNPEP locus in the genome of other species. Domains or motifs
were predicted with SMART (a Simple Modular Architecture Research Tool,
which allows the identification and annotation of genetically mobile
domains and the analysis of domain architectures; see
http://smart.embl-heidelberg.de/).

[0088] DNPEP is tandemly arranged with PTPRN in a conserved syntenic
region. The facts that PTPRN was clustered with RESP18 across mammalian
species, matched the sequence at the first 200 amino acids at the luminal
region, as well as expressed in the same subcellular location in islet
cells of the pancreas, prompted the hypothesis that the conserved
syntenic region of PTPRN may have additional genes sharing properties
including a similar expression pattern. Thus, the 163-kb PTPRN-containing
BAC clone (RP11-747C8) on human chromosome 2q35 was analyzed. Orthologs
of PTPRN, RESP18 and DNPEP were found clustered in human genome were also
present in the same order located on mouse chromosome 1. Such a syntenic
segment (PTPRN-RESP18-DNPEP in order) was found in all seven of the
mammalian species examined, including Homo sapiens (chromosome 2q35), Pan
troglodytes (Chr. 2b), Macaca mulatta (Chr. 12), Bos taurus (Chr. 2),
Canis lupus familiaris (Chr. 37), Rattus norvegicus (Chr. 9q33), and Mus
musculus (Chr. 1).

[0089] To determine if the syntenic block was evolutionarily conserved in
non-mammal animals, genomes in more species including Danio rerio,
Drosophila melanogaster, and C. elegans were searched and analyzed. The
ortholog of RESP18 was not found in any non-mammal species examined. The
ortholog of DNPEP was not found in the insect species of either
Drosophila melanogaster or Anopheles gambiae. However, ortholog genes of
PTPRN and DNPEP were found in all other four examined species including
Gallus gallus (Chr. 7), Xenopus tropicalis (Chr. location, unknown),
Danio rerio (Chr. 6) and C. elegans (Chr. III), suggesting that
PTPRN-DNPEP is a several million years old syntenic segment. It also
found that the cluster was duplicated in Danio rerio (Chr. 9 and Chr. 6).
However, the Dnpep gene on Chr. 9 of Danio rerio was missing. In C.
elegans, there are 17 other genes present in the region (less than 100
kb) between Ptprn (also known as ida-1) and Dnpep (e.g., F01F1.9). No
identical expression patterns were identified in the 17 genes according
to the Worm Database. Moreover, none of these 17 genes were syntenically
mapped to the PTPRN-DNPEP-containing regions in other species.

[0092] As shown in FIGS. 2A and 2B, PTPRN and RESP18 were both expressed
in islet cells with a stronger signal in the peripheral region. As shown
in FIG. 2C, results showed that DAP was also localized in peripheral
cells of islets. This specific pattern suggested that DAP was either
expressed in glucagon-positive alpha cells or somatostatin-positive delta
cells. Further analysis confirmed that DAP fluorescent signal was
completely colocalized with glucagon as detected by anti-glucagon
monoclonal antibody, as shown in FIGS. 2D and 2E, but not colocalized
with either insulin beta cells or somatostatin delta cells.

[0095] Expression levels in different brain regions were downloaded as xml
files and processed with Microsoft Excel 2007. Sagittal planes of the
spinal cord were taken at cervical spinal level (C1) for further analysis
of DNPEP, RESP18, and PTPRN expression data. To examine whether DNPEP
expression in neuronal tissues is similar to PTPRN and RESP18, the area
where messenger RNAs that encode DAP, RESP18 and PTPRN proteins are
expressed in adult (56 days) mouse brain tissues, using the ABA data sets
was analyzed. The probes used for in situ hybridization were 781-bp of
mouse DNPEP (nt 1058-1819, GenBank acc. no., NM--001110831), 591-bp
of mouse RESP18 (nt 60-650, GenBank acc. no., NM--009049), and
713-bp of mouse PTPRN (nt 1947-2659, GenBank acc. no., NM--008985),
respectively. The identities/similarities among these probes were low
(<40-45%) with no significance. However, the expression patterns of
Dnpep, Resp18 and Ptprn were very similar, present in various regions of
brain as shown in FIG. 3. Detailed analysis showed that higher expression
levels of Dnpep, Resp18 and Ptprn were in the midbrain, pons, medulla,
thalamus, main olfactory bulb, and hippocampus regions. Lower expression
levels of these genes were found in the subthalamic nucleus, hippocampal
region, cerebral cortex, caudate putamen, and cerebellum as indicated
respectively in FIG. 3.

[0096] Dnpep, Resp18 and Ptprn were expressed with similar patterns in all
twenty segments of the spinal cord from the cervical to lumber spinal
cord. FIG. 4 illustrates that the mRNAs of the three genes were similarly
distributed in the uppermost cervical segment of the spinal cord of adult
mice, mainly located in neuron-rich regions of gray matter.

Example 4

[0097] Immunoelectron microscopy: DAP was found in secretory granules.

[0099] Electron micrographs showed that DAP was associated with
lysosomal-like structures (FIG. 5A) as well as secretory granules in
islet alpha cells (FIG. 5B). Quantitative analysis of DAP particles per
square micrometer in 20 electron microscopic images revealed that there
were 84±13 particles around lysosome and 32±9 in secretory granules
comparing to 9±3 particles in the cytoplasm (mean±S.D.; P<0.01).
By double immunolabeling with rabbit antibody to DAP (5 nm colloidal gold
particles conjugated to goat anti-rabbit antibody) and mouse antibody to
glucagon (10 nm of colloidal gold conjugated with goat anti-mouse
antibody), we found that DAP was colocalized with glucagon in islet alpha
cells (FIG. 5C).

Example 5

[0100] DAP Enzymatic Activity

[0101] To examine whether the DAP aminopeptidase activity in cells was
reconciled with the subcellular distribution shown in the electron
microscopic analysis, DAP enzymatic activities in fractionated mouse
brain tissues was measured. Since DAP was found in secretory granules in
islet cells, it was assumed that DAP could be colocalized with vesicles
in neuronal cells of brain. Synaptosomes that contain secretory vesicles
and mitochondria of presynaptic terminal and postsynaptic membranes were
prepared from brain tissues by classical subcellular fractionation
techniques. Lysosome-, microsome-, and nuclei-enriched fractions were
also prepared for enzymatic activity analysis. Synaptosome that contains
vesicles was enriched as previously described. See, e.g., Bai et al.,
Subcell. Biochem., 43, 77-98 (2007).

[0102] Briefly, whole mouse brain was homogenized in buffer (0.32 M
sucrose, 20 mM HEPES, pH 7.4, with protease inhibitor cocktail, Sigma)
and centrifuged at 1000 g for 10 minutes to pellet the membrane fragments
and nuclei. The supernatant was then centrifuged at 17,000 g for 15
minutes to obtain the pellet-containing synaptosomes contaminated with
mitochondria and microsomes. This crude synaptosome fraction was further
purified by using a discontinuous sucrose density gradient consisting of
a 0.8 M sucrose layer on the top and a 1.2 M sucrose layer on the bottom.
The pure synaptosomal fraction can be obtained from the interface of 0.8
M and 1.2 M sucrose after 90 minutes centrifugation at 54,000 g. The
lysosome- or nuclei-containing fraction was then enriched with other
appropriate reagents (Instructions 89839 and 89841 respectively, Pierce,
Rockford, Ill., USA).

[0103] Membrane-bound DAP activity was measured as previously described in
the buffer of 50 Tris-HCl (pH 7.4), 1 mM MnCl2 and 0.125 mM
aspartyl-beta-NA. See, e.g., W. Wang et al., Mol. Biol. Evol., 24,
784-791 (2007). Relative fluorescence was converted into pmoles of
product using a standard curve, constructed with increasing
concentrations of beta-naphthylamine. The results were recorded as units
of aminopeptidase per mg protein of analyzed tissue. One unit of
aminopeptidase activity was the amount of enzyme that hydrolyzed 1 pmol
of aminoacyl-beta-naphthylamide per minute. Protein concentrations were
measured by Micro BCA Protein Assay (Pierce). Data was evaluated using
one-way analysis of the variance (ANOVA test), followed by the DMS test
for comparisons between more than two groups.

[0104] Data showed that the highest activities of DAP (˜400 units)
were detected in the synaptosomal and the lysosomal fractions as shown in
FIG. 6. Although the electron microscopic analysis did not show the
presence of DAP in ER and Golgi or nuclei, lower activities were also
found in the fractions of microsome (˜142 units) and nuclei
(˜57 units) as shown in FIG. 6. It cannot be excluded, however,
that the lower activities were attributed by small amount heterogenous
fractions of cytosol proteins that contained the DAP protein.

Example 6

[0105] Modulation of Angiotensin II Levels by DAP

[0106] DAP proteolytic activities are responsible for deleting the
N-terminal aspartic residues (Asp) of both Ang I (1-14)
(Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser) and Ang II
(1-8) (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe), which lead to the production of
Ang I* (des-Asp-Ang I) and Ang III (des-Asp-Ang II), respectively. Since
the angiotensin-renin system (ARS) is abundantly expressed in local islet
a cells and MIN6 cells, whether DAP expression modulates the Ang II
levels in islet cells was investigated.

[0107] The DAP gene was either knocked down with DAP RNAi construct or
overexpressed by transient transfection of DAP-pCMV in MIN6 cells.

[0108] The full-length DAP construct or the control vector was transfected
into MIN6 cells. After culturing for 48 hours, MIN6 cells and the culture
media were harvested for measurement of DAP and Ang II levels. Higher
expression level of DAP (2.1-fold±0.6) in DAP-pCMV-transfected MIN6
cells compared to that in the vector-transfected cells was detected by
Western blots. Ang II levels in MIN6 cell lysates and the cultured media
were measured after the transfection and compared to the control
transfected cells. Ang II-specific ELISA kit (Cayman Chemical) was used
for the measurements.

[0109] In the DAP-transfected cells, intracellular Ang II levels were 28
percent lower than that in vector-treated cells (22±4 pg/mg vs.
34±3 pg/mg protein respectively in mean±SEM, p<0.01) and the
extracellular Ang II levels were 35 percent lower than that in control
cells (22±3 pg/mg vs. 34±4 pg/mg protein respectively, p<0.01).
The data represented as percentages of controls are from at least three
independent experiments. The decrease of both intracellular and
extracellular Ang II levels in DAP transfected MIN6 cells, as shown in
FIG. 7, suggests an enhanced hydrolysis of Ang II.

[0111] The cells were transfected with 2 pg of Dnpep siRNA of SEQ ID NO.
13, non-silencing control siRNA, or a full-length of Dnpep cDNA (SEQ ID
NO. 12) in pCMV-SPORT6 (Open Biosystems, Huntsville, Ala.) by
electroporation (BioRad Laboratories, Berkeley, Calif.). A total of
1×105 of the cells was seeded into 6-well culture plates
coated with poly-L-lysine and cultured for 24 h. The transfection
efficiency was approximately 50 percent according to a co-transfected GFP
marker.

[0112] After 48 hours culture, cells and culture media were collected.
Real-time PCR (RT-PCR) was conducted with the primers (Assay ID Details:
Mm00497488_m1*) and the 7900HT Fast Real-Time PCR System (Applied
Biosystems, Foster City, Calif.). Western blots were performed using the
ECL Reagents (GE, Piscataway, N.J.).

[0113] In the DAP RNAi-transfected MIN6 cells, the endogenous DAP mRNA
level was substantially knocked down by 78 percent as shown by RT-PCR
analysis, and the DAP protein level decreased 70 percent compared to that
in control cells by Western blots. Consequently, the intracellular Ang II
levels increased up to 74 percent compared to the control cells (50±6
pg/mg vs. 28.7±4 pg/mg protein respectively, p<0.01) and
extracellular Ang II levels increased 57 percent compared to the control
cells (52±5.3 pg/mg vs. 33.1±4.2 pg/mg protein respectively,
p<0.01) (FIG. 7). The data represented as percentages of controls are
from at least three independent experiments. The results suggest that DAP
is a major hydrolase for Ang II conversion in islet cells.

[0114] Ang II levels were measured using the AssayMax Ang II ELISA Kit
(Cayman Chemical, Ann Arbor, Mich.). Briefly, standards or samples were
incubated in a 96-well plate with biotinylated anti-Ang II antibody for 2
h. After the washings, Streptavidin-Peroxidase Conjugate was added for 30
min. The final reaction was developed with the substrate chromogen and
read at 450 nm by an ELISA reader. Ang II levels in the samples were
calculated from an Ang II standard curve run with each assay. Values
(mean±SEM) are from at least three independent experiments. ANOVA with
Tukey's post hoc test was used for statistical analysis.

[0115] While the invention has been particularly described with specific
reference to particular processes and embodiments, it will be appreciated
that various alterations, modifications, and adaptations may be based on
the present disclosure, and are intended to be within the spirit and
scope of the invention as defined by the following claims.